'Warburg effect' controls tumor growth, bacterial, viral infections and immunity - Genetic deconstruction and therapeutic perspectives.

The evolutionary pressure for life transitioning from extended periods of hypoxia to an increasingly oxygenated atmosphere initiated drastic selections for a variety of biochemical pathways supporting the robust life currently present on the planet. First, we discuss how fermentative glycolysis, a primitive metabolic pathway present at the emergence of life, is instrumental for the rapid growth of cancer, regenerating tissues, immune cells but also bacteria and viruses during infections. The 'Warburg effect', activated via Myc and HIF-1 in response to growth factors and hypoxia, is an essential metabolic and energetic pathway which satisfies nutritional and energetic demands required for rapid genome replication. Second, we present the key role of lactic acid, the end-product of fermentative glycolysis able to move across cell membranes in both directions via monocarboxylate transporting proteins (i.e., MCT1/4) contributing to cell-pH homeostasis but also to the complex immune response via acidosis of the tumor microenvironment. Importantly lactate is recycled in multiple organs as a major metabolic precursor of gluconeogenesis and energy source protecting cells and animals from harsh nutritional or oxygen restrictions. Third, we revisit the Warburg effect via CRISPR-Cas9 disruption of glucose-6-phosphate isomerase (GPI-KO) or lactate dehydrogenases (LDHA/B-DKO) in two aggressive tumors (melanoma B16-F10, human adenocarcinoma LS174T). Full suppression of lactic acid production reduces but does not suppress tumor growth due to reactivation of OXPHOS. In contrast, disruption of the lactic acid transporters MCT1/4 suppressed glycolysis, mTORC1, and tumor growth as a result of intracellular acidosis. Finally, we briefly discuss the current clinical developments of an MCT1 specific drug AZ3965, and the recent progress for a specific in vivo MCT4 inhibitor, two drugs of very high potential for future cancer clinical applications.

Hazard Group 3 agent decontamination using hydrogen peroxide vapour in a class III microbiological safety cabinet.

AIMS: This study investigated the efficacy of hydrogen peroxide vapour (HPV) at inactivating hazard group 3 bacteria that have been presented dried from their growth medium to present a realistic challenge. METHODS AND RESULTS: Hydrogen peroxide vapour technology (Bioquell) was used to decontaminate a class III microbiological safety cabinet containing biological indicators (BIs) made by drying standard working suspensions of the following agents: Bacillus anthracis (Ames) spores, Brucella abortus (strain S99), Burkholderia pseudomallei (NCTC 12939), Escherichia coli O157 ST11 (NCTC 12079), Mycobacterium tuberculosis (strain H37Rv) and Yersinia pestis (strain CO92) on stainless steel coupons. Extended cycles were used to expose the agents for 90 min. The HPV cycle completely inactivated B. anthracis spores, B. abortus, B. pseudomallei, E. coli O157 and Y. pestis when BIs were processed using quantitative and qualitative methods. Whilst M. tuberculosis was not completely inactivated, it was reduced by 4 log10 from a starting concentration of 106 colony-forming units. CONCLUSIONS: This study demonstrates that HPV is able to inactivate a range of HG3 agents at high concentrations with associated organic matter, but M. tuberculosis showed increased resistance to the process. SIGNIFICANCE AND IMPACT OF THE STUDY: This publication demonstrates that HPV can inactivate HG3 agents that have an organic load associated with them. It also shows that M. tuberculosis has higher resistance to HPV than other agents. This shows that an appropriate BI to represent the agent of interest should be chosen to demonstrate a decontamination is successful.

Carbonic anhydrase XII expression is linked to suppression of Sonic hedgehog ligand expression in triple negative breast cancer cells.

Aberrant activity of the hedgehog (Hh) pathway is prevalent in pathologies such as cancer. Improved understanding of Hh activity in the aggressive tumor cell phenotype is being pursued for development of targeted therapies. Recently, we described a link between Hh activity and carbonic anhydrase XII (CAXII) expression. Extracellular facing CAs (IX/XII) are highly expressed in hypoxia, contribute to tumor pH regulation and are thus of clinical interest. Here we have extended the investigation of potential interactions between Hh activity and CAXII utilizing genomic disruption/knockout of either GLI1 (the main transcriptional factor induced with Hh activity) or CAXII in the triple negative breast cancer cell lines MDA-MB-231 and BT-549. Knockout of GLI1 and CAXII significantly decreased hallmarks of tumor aggressiveness including proliferation and migration. Most intriguingly, CAXII knockout caused a massive induction of the Sonic hedgehog (Shh) ligand expression (gene and protein). This novel finding indicates that CAXII plays a potential role in suppression of Shh and may act in a feedback loop to regulate overall Hh activity. Enhanced knowledge of these CA-Hh interactions in future studies may be of value in understanding this currently 'incurable' subclass of breast cancer.

Maternal anaemia and maternal, fetal, and neonatal outcomes in a prospective cohort study in India and Pakistan.

OBJECTIVE: To describe the association of maternal anaemia with maternal, fetal, and neonatal outcomes. DESIGN: Prospective cohort study. SETTING: Rural India and Pakistan. POPULATION: Pregnant women residing in the study catchment area. METHODS: We performed an analysis of a prospective pregnancy registry in which haemoglobin is commonly obtained as well as maternal, fetal, and neonatal outcomes for 42 days post-delivery. Women 40 years or older who delivered before 20 weeks or had a haemoglobin level of <3.0 g/dl were excluded. Our primary exposure was maternal anaemia, which was categorised in keeping with World Health Organization criteria based on a normal (≥11 g/dl), mild (>10-10.9 g/dl), moderate (7-9.9 g/dl) or severe (<7 g/dl). haemoglobin level. The primary maternal outcome was maternal death, the primary fetal outcome was stillbirth, and the primary neonatal outcome was neonatal mortality <28 days. RESULTS: A total of 92 247 deliveries and 93 107 infants were included, of which 87.8% were born to mothers who were anaemic (mild 37.9%, moderate 49.1%, and severe 0.7%). Maternal mortality (number per 100 000) was not associated with anaemia: normal 124, mild 106, moderate 135, and severe 325 (P = 0.64). Fetal and neonatal mortality was associated with severe anaemia: stillbirth rate (n/1000)-normal 27.7, mild 25.8, moderate 30.1, and severe 90.9; P < 0.0001; 28-day neonatal mortality (n/1000)-normal 24.7, mild 22.9, moderate 28.1, and severe 72.6 (P < 0.0001). Severe maternal anaemia was also associated with low birthweight (<2500 and <1500 g), preterm birth, and postpartum haemorrhage. CONCLUSION: Severe maternal anaemia is associated with higher risks of poor maternal, fetal, and neonatal outcomes but other degrees of anaemia are not. Interventions directed at preventing severe anaemia in pregnant women should be considered. TWEETABLE ABSTRACT: Severe maternal anaemia is associated with adverse fetal and neonatal outcomes in low/middle-income countries.

Is there an infection risk when playing drums contaminated with Bacillus anthracis?

AIMS: This study aims to investigate the aerosol release of a Bacillus anthracis spore surrogate from two different types of drums while playing, by; (i) quantifying the number of spores aerosolized during playing; (ii) investigating spore recovery from drums over long time periods, and (iii) measuring differences between (i) and (ii) for two different drums types. METHODS AND RESULTS: Two African drums were contaminated with Bacillus atrophaeus spores then sampled and played by hand over a number of days. During playing three air samplers were used to collect any aerosols generated, the choice of air samplers (Casella slit sampler, all glass impinger and six-stage Andersen sampler) allowed for characterization of the aerosols produced. CONCLUSIONS: Spore contamination of drums was found to be long-lasting with a small percentage of the spores being detached and aerosolized during drumming. The results of these studies have been used for a quantitative risk assessment of playing drums contaminated with B. anthracis spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This demonstrates that the risk of inhalational exposure while playing drums contaminated with the levels linked to the US and UK cases is very low and that the resulting cases of inhalational anthrax can be explained by being unusual events involving highly susceptible persons.

Expression of the hypoxia-inducible monocarboxylate transporter MCT4 is increased in triple negative breast cancer and correlates independently with clinical outcome.

BACKGROUND: (18)Fluor-deoxy-glucose PET-scanning of glycolytic metabolism is being used for staging in many tumors however its impact on prognosis has never been studied in breast cancer. METHODS: Glycolytic and hypoxic markers: glucose transporter (GLUT1), carbonic anhydrase IX (CAIX), monocarboxylate transporter 1 and 4 (MCT1, 4), MCT accessory protein basigin and lactate-dehydrogenase A (LDH-A) were assessed by immunohistochemistry in two cohorts of breast cancer comprising 643 node-negative and 127 triple negative breast cancers (TNBC) respectively. RESULTS: In the 643 node-negative breast tumor cohort with a median follow-up of 124 months, TNBC were the most glycolytic (≈70%), followed by Her-2 (≈50%) and RH-positive cancers (≈30%). Tumoral MCT4 staining (without stromal staining) was a strong independent prognostic factor for metastasis-free survival (HR=0.47, P=0.02) and overall-survival (HR=0.38, P=0.002). These results were confirmed in the independent cohort of 127 cancer patients. CONCLUSION: Glycolytic markers are expressed in all breast tumors with highest expression occurring in TNBC. MCT4, the hypoxia-inducible lactate/H(+) symporter demonstrated the strongest deleterious impact on survival. We propose that MCT4 serves as a new prognostic factor in node-negative breast cancer and can perhaps act soon as a theranostic factor considering the current pharmacological development of MCT4 inhibitors.

Evaluation of a method to detect Mycobacterium bovis in air samples from infected Eurasian badgers (Meles meles) and their setts.

Environmental air sampling was evaluated as a method to detect the presence of M. bovis in the vicinity of infected badgers and their setts. Airborne particles were collected on gelatine filters using a commercially available air sampling instrument and tested for the presence of M. bovis using bacteriological culture and real-time PCR. The sensitivity of bacteriological culture was broadly similar to that of real-time PCR when testing samples artificially spiked with M. bovis. Sampling was undertaken from directly under the muzzles of badgers which had been experimentally infected with M. bovis (37 samples), within enclosures housing the experimentally infected animals (50 samples), and in the vicinity of setts with resident infected wild badgers (52 samples). The methods employed did not detect M. bovis from either infected badgers or artificial or natural setts known to contain infected animals. However, samples taken at four of the six natural setts were positive for Mycobacterium gordonae.

Why whales are big but not bigger: Physiological drivers and ecological limits in the age of ocean giants.

The largest animals are marine filter feeders, but the underlying mechanism of their large size remains unexplained. We measured feeding performance and prey quality to demonstrate how whale gigantism is driven by the interplay of prey abundance and harvesting mechanisms that increase prey capture rates and energy intake. The foraging efficiency of toothed whales that feed on single prey is constrained by the abundance of large prey, whereas filter-feeding baleen whales seasonally exploit vast swarms of small prey at high efficiencies. Given temporally and spatially aggregated prey, filter feeding provides an evolutionary pathway to extremes in body size that are not available to lineages that must feed on one prey at a time. Maximum size in filter feeders is likely constrained by prey availability across space and time.

Clinical field study of the safety and efficacy of spinosad chewable tablets for controlling fleas on dogs.

Preliminary studies showed spinosad to be rapidly effective and safe in controlling fleas on dogs. To validate these studies, a clinical trial was undertaken using 470 flea-infested client-owned dogs allocated to receive three monthly treatments with either beef-flavored chewable spinosad tablets (30-60 mg/kg) or selamectin applied according to label instructions. Flea counts 15 days after enrollment were reduced by 98.6% and 90.9% for spinosad- and selamectin-treated dogs, respectively; at 90 days, flea count reductions were 99.9% and 98.9%, respectively. Compared with baseline, all flea reductions were significant (P < .001) for both products and spinosad was significantly (P ≤ .0172) more effective than selamectin at each postenrollment flea count.

Electrical transport measurements of thin film samples under high hydrostatic pressure.

We present a method to perform electrical measurements of epitaxial films and heterostructures a few nanometers thick under high hydrostatic pressures in a diamond anvil cell (DAC). Hydrostatic pressure offers the possibility to tune the rich landscape of properties shown by epitaxial heterostructures, systems in which the combination of different materials, performed with atomic precision, can give rise to properties not present in their individual constituents. Measuring electrical conductivity under hydrostatic pressure in these systems requires a robust method that can address all the challenges: the preparation of the sample with side length and thickness that fits in the DAC setup, a contacting method compatible with liquid media, a gasket insulation that resists high forces, as well as an accurate procedure to place the sample in the pressure chamber. We prove the robustness of the method described by measuring the resistance of a two dimensional electron system buried at the interface between two insulating oxides under hydrostatic conditions up to ∼5 GPa. The setup remains intact until ∼10 GPa, where large pressure gradients affect the two dimensional conductivity.

Microbiological problems and biofilms associated with Mycobacterium chimaera in heater-cooler units used for cardiopulmonary bypass.

The role of heater-cooler units (HCUs) in the transmission of Mycobacterium chimaera during open heart surgery has been recognized since 2013. Subsequent investigations uncovered a remarkable global outbreak reflecting the wide distribution of implicated devices. HCUs are an essential component of cardiopulmonary bypass operations and their withdrawal would severely affect capacity for life-saving cardiac surgery. However, studies have demonstrated that many HCUs are contaminated with a wide range of micro-organisms, including M. chimaera and complex biofilms. Whole genome sequencing of M. chimaera isolates recovered from one manufacturer's HCUs, worldwide, has demonstrated a high level of genetic similarity, for which the most plausible hypothesis is a point source contamination of the devices. Dissemination of bioaerosols through breaches in the HCU water tanks is the most likely route of transmission and airborne bacteria have been shown to have reached the surgical field even with the use of ultraclean theatre ventilation. Controlling the microbiological quality of the water circulating in HCUs and reducing biofilm formation has been a major challenge for many hospitals. However, enhanced decontamination strategies have been recommended by manufacturers, and, although they are not always effective in eradicating M. chimaera from HCUs, UK hospitals have not reported any new cases of M. chimaera infection since implementing these mitigation strategies. Water safety groups in hospitals should be aware that water in medical devices such as HCUs may act as a vector in the transmission of potentially fatal water-borne infections.

Production of monoclonal antibody alpha Pro3 recognizing a human prostatic carcinoma antigen.

The monoclonal antibody alpha Pro3 recognizes an antigen concentrated in human primary prostatic carcinoma tissue removed surgically. Although the antigen was detectable in extracts of human normal and malignant nonprostatic tissue, as well as in benign prostate tissue, quantitative absorption analysis revealed a substantially greater quantity of the antigen in malignant prostate tissue. The antigen recognized by alpha Pro3 in primary prostatic carcinoma has an apparent nonreduced molecular weight of 175,000 and an apparent subunit molecular weight of 54,000. This antigen, p54, appears to be present on the surface of cultured prostatic tumor cells of the PC-3 cell line, but its location in vivo has not been defined. Successful competition between alpha Pro3 and prostatic carcinoma patient serum immunoglobulin for a Mr 54,000 antigen (reduced molecular weight) present in prostatic carcinoma tissue extract suggests that p54 may play a significant role in the immunobiology of prostatic carcinoma. alpha Pro3 has potential as a sensitive probe for an antigen relevant to human tumor biology.

Retinal toxicity during pegylated alpha-interferon therapy for chronic hepatitis C: a multifocal electroretinogram investigation.

BACKGROUND: Ocular side-effects in the form of retinal ischaemia and haemorrhages have been reported in patients undergoing standard alpha-interferon therapy. AIM: To assess the ocular impact of therapy with sustained release pegylated alpha-2a interferon (Pegasys) for chronic hepatitis C. METHODS: Ten patients receiving Pegasys and ribavirin and 10 healthy volunteers were recruited. Patients underwent full ophthalmic investigations and multifocal electroretinogram testing at baseline, at regular intervals during treatment and post-treatment. The multifocal electroretinogram maps retinal function. Responses were compared with sequential recordings from healthy volunteers. RESULTS: All patients had normal clinical ophthalmic investigations at baseline. During therapy a single patient experienced central visual disturbance lasting 24 h with no prolonged ill effect. No other patient was aware of any change in vision. Fundal abnormalities appeared in five patients during treatment. The multifocal electroretinogram showed reductions in retinal function in five patients. Nine of 10 patients exhibited abnormalities on at least one multifocal electroretinogram or fundoscopic investigation. CONCLUSIONS: Subclinical retinal toxicity during anti-viral therapy with pegylated alpha-interferon and ribavirin was frequent in this study and it suggests that patients should be warned of this risk and monitored during therapy.

Determining the performance of a commercial air purification system for reducing airborne contamination using model micro-organisms: a new test methodology.

The performance of a duct-mounted air disinfection system, designed to reduce airborne pathogens in the hospital environment, was determined using a new testing methodology. The methodology places the equipment in a test duct, a microbial aerosol is generated and then sampled simultaneously before and after the test system. This allows a percentage efficiency value to be calculated. The air disinfection system is a novel chemical-coated filter and ultraviolet (UV) radiation air purification system, operating at a flow rate of 500 m(3)/h, against aerosols of MS2 phage and Mycobacterium vaccae (surrogates of viral and mycobactericidal pathogens). A three UV lamp system was effective against airborne phages, removing an average of 97.34% of the aerosolized challenge. With the UV component switched off, the average efficiency dropped to 61.46%. This demonstrates that the chemical-coated filter component plays a more significant role than the UV radiation in destroying phages. When six UV lamps were used, the system was able to remove mycobacteria with an efficiency exceeding 99.99%. This test methodology can be used to assess manufacturers' claims of efficacy of equipment against airborne micro-organisms in the hospital environment.

Spatial localization in esotropia: does extraretinal eye position information change?

PURPOSE: To investigate the accuracy of spatial localization in children with a specific type of convergent strabismus, fully accommodative esotropia. METHODS: Two groups of children, with right and left fully accommodative esotropia, respectively, pointed at targets located centrally and eccentrically on a computer touchscreen without being able to see their hands. The size and the direction of the horizontal pointing responses were recorded under two conditions: when their eyes were aligned (wearing spectacles) and when they were squinting (not wearing spectacles). A group of children without strabismus but with hypermetropia were assessed as controls. RESULTS: For both fully accommodative groups, the pointing responses to the central target shifted in the direction of the nonsquinting eye when deviations were manifest. No difference was found for the eccentric targets. No difference was found for the hypermetropia group with any target. CONCLUSIONS: These results are consistent with an alteration in the extraretinal eye position information (efference copy, extraocular muscle proprioception, or both) that is used in spatial localization.

Spatial localization after different types of retinal detachment surgery.

PURPOSE: To compare the effect on spatial localization of two different forms of surgery for primary rhegmatogenous retinal detachment. METHODS: Two groups of 30 patients (one group undergoing conventional external scleral-buckling procedures, the other undergoing vitrectomy procedures) were recruited. They pointed at targets appearing on a computer touchscreen without being able to see their hands, while viewing targets with the non-surgically treated eye. The sizes of the horizontal pointing errors were recorded on three separate occasions: before surgery, on the first postoperative day, and approximately 10 days later. RESULTS: On the first postoperative day a significant change in localization of 2.9 +/- 0.9 degrees [SD]) was observed in the scleral-buckling group, compared with 1.3 +/- 0.6 degrees in the vitrectomy group. These changes resolved by the second postoperative assessment. CONCLUSIONS: These results, particularly in patients in the scleral-buckling group in whom greater manipulation of the extraocular muscles inevitably occurs, are consistent with an alteration in the extraretinal eye position information that is used in spatial localization. This is likely to be a consequence of modified efference copy and/or extraocular muscle proprioception.

Beta-galactosidase deficiency: studies of two patients with prolonged survival.

Two adults with beta-galactosidase deficiency were studied. Differences in a number of beta-galactosidase parameters (pH optima, heat denaturation, NaCl kinetics) were noted between the patients. Differences were also noted in beta-galactosidase electrophoretic mobilities and urinary oligosaccharides; however, there was no complementation in cell fusion studies. It is suggested that these two patients have different primary mutations at the beta-galactosidase locus which are probably structural in nature.

Real-time polymerase chain reaction with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes.

We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]-restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% +/- 25%) than did nonhomozygotes (n = 340; 51.4% +/- 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 +/- 0.26), 1 (n = 84; APCR = 1.61 +/- 0.13), or 2 (n = 5; APCR = 1.16 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations.

Storage and transportation of lymphoid tissue for immunophenotyping.

Immunoperoxidase staining of frozen sections is a cost-effective technic for immunophenotyping cells of lymphoid tissue. Because this procedure is not performed in many institutions, a simple method to transport fresh tissue to centers performing these studies is required. Tissues in saline at refrigerator temperature may be successfully transported. In addition, in order to minimize laboratory expenses, lymphoid tissue can be kept refrigerated in saline until permanent sections are examined and immunodiagnostic procedures become necessary. In this study reproducible immunophenotyping of 12 samples of lymphoid tissue stored up to seven days was achieved.